Currently, embryo selection for transfer relies on morphological criteria evaluation with optical microscopy in Assisted Reproductive Technology (ART). The parameters classically involved in embryo selection are: i) morphology and fertilization of oocyte having generated embryo, ii) number and size of blastomeres, iii) cytoplasmic appearance of blastomeres, iv) fragmentation rate and v) multinucleation presence. This morphological approach is based exclusively on subjective observations of embryo morphology, dependent operator and shows limitations to predict successful pregnancy in ART (Guerif et al., 2007). Indeed, currently, 85% of embryos obtained in vitro and selected for replacement on morphological criteria lead to implantation failures. This result is completely disappointing and suggests that the limited 2D-visualization of human oocyte and embryo with optical microscopy lacks accuracy. Therefore, there is a reliable need in ART to improve human oocyte and embryo observation during their early preimplantation development and subsequently increase ART success.
Recently, a new method based on time-lapse imaging has been developed for the acquisition of embryo morphokinetic data to help such selection (Meseguer et al., 2011; Herrero and Meseguer, 2013). Nevertheless, time-lapse systems do not improve embryo morphology evaluation compared to optical microcopy since embryos contained in individual wells are observed only in 7 focal planes. Indeed, a major limitation of these systems is the inability to rotate or roll spherical oocyte and embryos making it difficult to assess morphology especially in the case of a high fragmentation rate or blastocyst stage. Moreover, regular purchase images requires light embryo. Even if the light is relatively short in these systems, an exposure time as short as possible associated with a wavelength longer closely as possible should be preferred.
The use of 3D technologies represents an innovative and attractive way in ART to develop new precious tools for studying human oocyte and early embryo and subsequently for better understanding the ART failures in the daily practice. Indeed, it seems very difficult to analyze in detail a spherical biological structure, such as human oocyte and embryo, only in 2D-planes. The constraints of the optical microscopy force us limit ourselves in human oocyte and embryo observation and 3D imaging methods could allow a sophisticated morphological assessment like in mouse model (Nieman et al., 2011). Other numerous morphological parameters could be studied with 3D technologies such as 3D pronuclei position in oocyte, 3D fragmentation repartition, 3D blastomeres cleavage axis or 3D cells repartition in the embryo. The main aim of the improvement of human oocyte and embryo observation by 3D technologies is improving ART success.
In the past years, 3D reconstructions of mouse embryos required dissections to obtain a series of histological and fixed sections in order to study embryo anatomical organization and characterize mouse morphological phenotypes (Kaufman et al., 1997; Wong et al., 2012; Wong et al., 2014). Nowadays, one of the challenges of the optical imaging is to observe in 3D the biological structures and organisms in compatible conditions for the pursuit of their development. Therefore, recently, new technologies in optical imaging field, have been developed to perform sections of viable embryos in some species and to allow a non-invasive 3D reconstruction of these embryos.
Among these new optical imaging systems, the optical coherence tomography (OCT) is a non-invasive emerging technique for biological media with micrometer-scale resolution used principally in ophthalmology. A new and original approach of OCT called full-field OCT has been proposed recently by a research team and is based on white-light interference microscopy. Full-field OCT allows to obtain tomographic pictures by combination of interferometric images recorded by a detector array such as CCD camera. Compared to conventional OCT, full-field OCT acquires tomographic images in transverse orientation with ultrahigh resolution (˜1 μm) using a simple halogen lamp. Interestingly, the performance of this technology has been used in embryology and developmental biology for 3D imaging of ex-vivo specimens. Indeed, 3D reconstructions of mouse embryo are easy performed with the same resolution than in standard histological method. Moreover, image acquisitions are fast and do not require sample preparation and dissection in thin sections after fixation compared to histological sections.
Light sheet microscopy including Selective Plane Illumination Microscopy (SPIM) is also a new technique in which the illuminated plane in a sample is the only being imaged, associated with a virtual elimination of background signal and a drastic reduction of the amount of light required to explore the sample. Indeed, this technique reduces phototoxic effects in live samples and allows to see biological structures in 3D, in live and in real time without harmful effects and damaging on them (Huisken et al., 2004, Huisken, 2012). This concept is based on illuminating the sample only with thin slices of light in a focal plane, and photo bleaching is decreased to a minimum. Therefore, light sheet microscopy constitutes an ideal tool for non-destructive imaging of fragile or viable samples. Regarding to embryology and development biology applications, light sheet microscopy (including SPIM) appears to be an attractive tool to image 3D embryos at high resolution with high acquisition speed while being minimally invasive. 3D early zebrafish embryo and 3D embryonic development of Drosophila melanogaster reconstructions illustrate the potential of SPIM technique in developmental biology (Huisken et al., 2004; Keller et al., 2008; Huisken and Stainier, 2009; Weber and Huisken, 2011; Kaufmann et al., 2012; Krzic et al., 2012, Huisken, 2012).
Therefore, optical microscopy allows performing 3D reconstructions and printing of mouse or human embryos from several focal planes. Moreover, OCT and light sheet microscopy (including SPIM) represent the best attractive innovations to develop non-invasive image acquisitions of human oocyte and embryo for 3D reconstructions and printing. The applications of these new optical imaging methods remains to be tested in human embryology for a sophisticated observation of spherical oocyte and embryo obtained in vitro.
Furthermore, several research teams have proposed an atlas book containing 3D mouse embryo reconstructions using magnetic resonance imaging but most of the time, they did not studied embryos at the early stages. The use of micro magnetic resonance imaging (μMRI) for 3D human oocyte and embryo reconstruction could be also an attractive way for oocyte and embryo morphology assessment, but nowadays, the image spatial resolution is not sufficient even at high field (even at 9.4 T).
Recently, the emergence of 3D printing and its application have expanded in human medicine (Rengier et al., 2010; Hespel et al., 2014). Indeed, these recent technological advances allow to create complex structures such as human cartilage tissue, heart valves, bone models or cranioplasty implant using 3D printer (Kim et al., 2012; Visser et al., 2013; Cui et al., 2014; Nakayama et al., 2014; Unger et al., 2014. Hochman et al., 2014; Tan et al., 2014). Like in surgery, one recent study reported the reproduction of zebrafish embryos and larvae models using a 3D printer (Masselink et al., 2014). Therefore, the 3D printing of human oocyte and embryo could allow the creation of several models of a more realistic nature, representing real oocyte and embryo development. Moreover, these human oocyte and embryo models may represent informative, pedagogic training and updating tools for embryologist staff, patients and students.